Human blastocyst-derived stem (hBS) cells have the unique ability to differentiate into derivatives of all three germ layers. This characteristic turns them into an exceptional tool in the field of toxicology as they can serve as a “cell factory” for functional cells (Moon S Y, et al, Mol Ther 13(1):514, 2006). Moreover, effects of compounds interacting during the process of hBS cell differentiation can be detected which makes them especially valuable in the field of developmental toxicology.
As the new European Chemicals Policy (REACH) will come into effect in 2007, toxicological information is required for more than 30.000 chemicals manufactured or imported in volumes above 1 ton annually (Anon, 2007). Consequently, around 3.9 million additional test animals will potentially be used and the costs to industry are estimated to be around 1.5 Billion Euro, of which 32% are attributed alone to developmental toxicity studies (RPA, 2002). Therefore, in vitro developmental toxicity tests are urgently needed. In addition, the pharmaceutical industry faces the demand for high throughput in vitro toxicity tests as reliable toxicological data for novel drug candidates have to be generated as early as possible in the development phase. The reduction of the high attrition rates due to incorporation of early in vitro toxicity screenings would reduce the associated costs enormously. Moreover, a number of substances are known to display significant inter-species differences and lead to severe malformations in humans but not distinctly in mice or rats, e.g. 13-cis retinoic acid (Isotretinoin) that is used in the treatment of severe acne (Accutane, Roche) and the sedative and anti-inflammatory drug thalidomide (Contergan) (Gilbert, 2003). Thus, human relevant developmental toxicity tests are required.
One of the most promising in vitro embryotoxicity tests to date is the validated embryonic stem cell test (EST), which employs murine embryonic stem (mES) cells to assess the embryotoxic potential of chemicals. The EST takes the different sensitivities of mES cells and murine fibroblasts to embryotoxicants into account. In addition, the differentiation of mES cells into functional cardiomyocytes serves as a toxicological endpoint (Genschow, 2004). However, the EST still aims to predict human toxicity in an animal system.
The use of human embryonic stem cells in a developmental toxicity test could provide reliable, human relevant data that add value to existing toxicity tests for safety assessment of drugs and chemicals. However, the application of hBS cells in toxicity testing is challenging as these cells require complex handling techniques. For example, hBS cells need to be seeded in cell aggregates instead of single cells to ensure their growth and show variable attachment capacities to surfaces that results in high variances. Additionally, the population-doubling time of hBS cells is with 36 hours significantly longer than that of mES cells with 12 hours. Another important difference between mouse and human BS cells are their culturing requirements. To maintain mES cells in an undifferentiated state the addition of leukaemia inhibitory factor (LIF) to the culture medium is sufficient. hBSC lines, however, are cultured on a mouse or human feeder layer, which appears to be the most reliable way to maintain cells stably in the undifferentiated state. Much work is being done to find feeder-free culture systems but these are at an early stage of development (Stacey et al, 2006)
The present invention represents a toxicity assay based on hBS cells for prediction of human toxicity, such as developmental toxicity and cytotoxicity. The present invention shows a great advantage over mES cells being able to deliver human relevant data that help identifying human teratogens of which some are known to display inter-species differences as for example 13-cis retinoic acid. Such a toxicity test would have the potential to be part of a testing strategy for the detection of human relevant developmental toxicants.